TY - JOUR
T1 - Production of nuclear transfer-derived piglets using porcine fetal fibroblasts transfected with the enhanced green fluorescent protein
AU - Hyun, Sanghwan
AU - Lee, Gabsang
AU - Kim, Daeyoung
AU - Kim, Hyesoo
AU - Lee, Sohyun
AU - Nam, Donghyun
AU - Jeong, Yeonwoo
AU - Kim, Sue
AU - Yeom, Soocheong
AU - Kang, Sungkeun
AU - Han, Jaeyong
AU - Lee, Byeongchun
AU - Hwang, Woosuk
PY - 2003/9/1
Y1 - 2003/9/1
N2 - A system for somatic cell nuclear transfer (SCNT) was developed and led to the successful production of GFP-transfected piglets. In experiment 1, two groups of SCNT couplets reconstructed with porcine fetal fibroblasts (PFF) and enucleated sow (S) or gilt oocytes (G): 1) received a simultaneous electrical fusion/activation (S-EFA or G-EFA groups), or 2) were electrically fused followed by activation with ionomycin (S-EFIA or G-EFIA groups), or 3) were subjected to electrical fusion and subsequent activation by ionomycin, followed by 6-dimethylaminopurine treatment (S-EFIAD or G-EFIAD groups). The frequency of blastocyst formation was significantly higher in S-EFA (26%) compared with that observed in the other experimental groups (P < 0.05), but not with S-EFIA (23%). Sow oocytes yielded significantly higher cleavage frequencies (68%-69%) and total cell numbers of blastocysts when compared with gilt oocytes, regardless of fusion/activation methods (P < 0.05). However, the ratio of inner cell mass (ICM)/total cells in G-EFA and S-EFA was significantly lower than in the other groups (P < 0.05). In experiment 2, SCNT couplets reconstructed with PFF cultured in the presence or absence of serum and enucleated sow oocytes were subjected to EFA. There were no effects of serum starvation on cell-cycle synchronization, developmental competence, total cell numbers, and ratio of ICM/total cells. In experiment 3, SCNT couplets reconstructed with PFF transfected with an enhanced green fluorescence protein (EGFP) gene using FuGENE-6 and enucleated sow oocytes were subjected to EFA and cultured for 7 days. Expression frequencies of GFP gene during development were 100%, 78%, 72%, 71%, and 70% in fused, two-cell, four to eight cells, morulae, and blastocysts, respectively. In experiment 4, SCNT embryos derived from different recipient cytoplasts (sows or gilts) and donor karyoplasts (PFF or GFP-transfected) were subjected to EFA and transferred to the oviducts of surrogates. The pregnancy rates in SCNT embryos derived from sow oocytes (66%-69%) were higher than those with gilt oocytes (23%-27%) regardless of donor cell types. One live offspring from GFP-SCNT embryos and two from PFFSCNT embryos were delivered. Microsatellite analysis confirmed that the clones were genetically identical to the donor cells and polymerase chain reaction (PCR) from genomic DNA of cloned piglets and subsequent southern blot analysis confirmed the integration of EGFP gene into chromosomes.
AB - A system for somatic cell nuclear transfer (SCNT) was developed and led to the successful production of GFP-transfected piglets. In experiment 1, two groups of SCNT couplets reconstructed with porcine fetal fibroblasts (PFF) and enucleated sow (S) or gilt oocytes (G): 1) received a simultaneous electrical fusion/activation (S-EFA or G-EFA groups), or 2) were electrically fused followed by activation with ionomycin (S-EFIA or G-EFIA groups), or 3) were subjected to electrical fusion and subsequent activation by ionomycin, followed by 6-dimethylaminopurine treatment (S-EFIAD or G-EFIAD groups). The frequency of blastocyst formation was significantly higher in S-EFA (26%) compared with that observed in the other experimental groups (P < 0.05), but not with S-EFIA (23%). Sow oocytes yielded significantly higher cleavage frequencies (68%-69%) and total cell numbers of blastocysts when compared with gilt oocytes, regardless of fusion/activation methods (P < 0.05). However, the ratio of inner cell mass (ICM)/total cells in G-EFA and S-EFA was significantly lower than in the other groups (P < 0.05). In experiment 2, SCNT couplets reconstructed with PFF cultured in the presence or absence of serum and enucleated sow oocytes were subjected to EFA. There were no effects of serum starvation on cell-cycle synchronization, developmental competence, total cell numbers, and ratio of ICM/total cells. In experiment 3, SCNT couplets reconstructed with PFF transfected with an enhanced green fluorescence protein (EGFP) gene using FuGENE-6 and enucleated sow oocytes were subjected to EFA and cultured for 7 days. Expression frequencies of GFP gene during development were 100%, 78%, 72%, 71%, and 70% in fused, two-cell, four to eight cells, morulae, and blastocysts, respectively. In experiment 4, SCNT embryos derived from different recipient cytoplasts (sows or gilts) and donor karyoplasts (PFF or GFP-transfected) were subjected to EFA and transferred to the oviducts of surrogates. The pregnancy rates in SCNT embryos derived from sow oocytes (66%-69%) were higher than those with gilt oocytes (23%-27%) regardless of donor cell types. One live offspring from GFP-SCNT embryos and two from PFFSCNT embryos were delivered. Microsatellite analysis confirmed that the clones were genetically identical to the donor cells and polymerase chain reaction (PCR) from genomic DNA of cloned piglets and subsequent southern blot analysis confirmed the integration of EGFP gene into chromosomes.
KW - Early development
KW - Embryo
KW - Gamete biology
KW - Ovary
UR - http://www.scopus.com/inward/record.url?scp=10744229521&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=10744229521&partnerID=8YFLogxK
U2 - 10.1095/biolreprod.102.014886
DO - 10.1095/biolreprod.102.014886
M3 - Article
C2 - 12773429
AN - SCOPUS:10744229521
SN - 0006-3363
VL - 69
SP - 1060
EP - 1068
JO - Biology of reproduction
JF - Biology of reproduction
IS - 3
ER -